We examined the role of individual integrins in promoting human keratinocyte migration. In short-term assays on collagen type I- or fibronectin-coated substrates, migration was blocked by antibody to the α2 integrin and the α5 integrin, respectively. Unexpectedly, antibodies to integrin α3 also significantly inhibited cell locomotion on both ligands. Time-course immunofluorescence staining revealed that keratinocyte migration was accompanied by deposition of endogenous laminin 5. Since α3β1 is a known receptor for this ligand, this observation suggested that migrating keratinocytes use freshly deposited laminin 5 in locomotion. Indeed, further investigation showed that anti-laminin 5 blocking antibodies effectively inhibited keratinocyte motility on both collagen and fibronectin substrates. Furthermore, cell migration on laminin 5-coated substrates was blocked by both anti-α3 and anti-laminin 5 antibodies. Laminin 5 did not appear important in the initial attachment of keratinocytes, since adhesion of cells to collagen type I- or fibronectin-coated surfaces was not blocked by antibody to α3 integrin or to laminin 5, but could be inhibited by antibody to α2 or α5, respectively. Using anin vitrowound assay, blocking antibodies to α3 integrin and to laminin 5 also blocked reepithelization of the denuded monolayer. These results show that α3β1 integrin plays an important role in the migration of keratinocytes via their interaction with laminin 5. Furthermore, they suggest that cell migration is dependent not only on exogenous ligands but, importantly, on endogenously secreted laminin 5. Finally, the data are consistent with our earlier finding that laminin 5 is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healingin vivo[1].